Genome-Enabled Analysis of Population Dynamics and Virulence-Associated Loci in the Oat Crown Rust Fungus Puccinia coronata f. sp. avenae.

Puccinia coronata f. sp. avenae (Pca) is an important fungal pathogen causing crown rust that impacts oat production worldwide. Genetic resistance for crop protection against Pca is often overcome by the rapid virulence evolution of the pathogen. This study investigated the factors shaping adaptive evolution of Pca using pathogen populations from distinct geographic regions within the United States and South Africa. Phenotypic and genome-wide sequencing data of these diverse Pca collections, including 217 isolates, uncovered phylogenetic relationships and established distinct genetic composition between populations from northern and southern regions from the United States and South Africa. The population dynamics of Pca involve a bidirectional movement of inoculum between northern and southern regions of the United States and contributions from clonality and sexuality. The population from South Africa is solely clonal. A genome-wide association study (GWAS) employing a haplotype-resolved Pca reference genome was used to define 11 virulence-associated loci corresponding to 25 oat differential lines. These regions were screened to determine candidate Avr effector genes. Overall, the GWAS results allowed us to identify the underlying genetic factors controlling pathogen recognition in an oat differential set used in the United States to assign pathogen races (pathotypes). Key GWAS findings support complex genetic interactions in several oat lines, suggesting allelism among resistance genes or redundancy of genes included in the differential set, multiple resistance genes recognizing genetically linked Avr effector genes, or potentially epistatic relationships. A careful evaluation of the composition of the oat differential set accompanied by the development or implementation of molecular markers is recommended. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Rust Caused by Phakopsora nishidana on Creeping Fig, Ficus pumila, in South Africa.

Phakopsora nishidana has recently been reported as the causal organism of rust on edible fig, Ficus carica in South Africa (SA) (Boshoff et al. 2022). This contradicted reports by Doidge (1927, 1950) and Verwoerd (1929) who listed Cerotelium fici as the causal organism of the disease in SA. Similarities in urediniospore morphology and differing taxonomic interpretations most likely contributed to the use of both pathogen names as the causal agents for fig rust (Boshoff et al. 2022; Padamsee and McKenzie 2024). In January 2023 rust was commonly observed on nursery specimens of creeping fig, Ficus pumila, a popular evergreen and fast-growing garden plant in SA. Ficus pumila is native to southern China, Indochina, and eastern Asia but has been introduced to many countries worldwide (https://powo.science.kew.org/ accessed 20 March 2024). The F. pumila rust isolate collected in Somerset West (34°07'00.70"S, 18°52'41.75"E; Western Cape (WC) province) was identified using the sequenced 5.8S rRNA-ITS2-28S rRNA locus. When used in a BLAST analysis, this F. pumila isolate (GenBank accession number OR835538) shared the best homology with the Vermont P. nishidana isolate (34°24'33.37"S, 19°08'59.24"E) (MZ047090; e-value 0.0, 99.7% identity, 1400/1404 bp) reported on edible fig by Boshoff et al. (2022). Other P. nishidana isolates with excellent homology were KY764080 (e-value 0.0, 99.8% identity, 1012/1014 bp), KY764081 (e-value 0.0, 99% identity, 882/885 bp) and MF580676 (e-value 0.0, 99.3% identity, 981/988 bp). Accessions were used in a phylogenetic study that included four other rust samples collected from F. carica trees in Somerset West, WC (OR835534; 34°03'37.26"S, 18°51'02.18"E), Onrus River, WC (OR835535; 34°24'43.10"S, 19°09'57.99"E), Elgin, WC (OR835536; 34°14'31.37"S, 19°03'05.38"E), and Bloemfontein, Free State province (OR835537; 29°05'05.1"S 26°09'09.5"E). Reference sequences were as described in Boshoff et al. (2022). Despite limited variation, all six South African isolates, including the F. pumila isolate, grouped with the three P. nishidana reference isolates, but separate from the three P. myrtacearum isolates collected from eucalyptus trees (Maier et al. 2016). Uredinia on F. pumila leaves were mostly hypophyllous and surrounded by a halo of brown, necrotic tissue. On the upper leaf surface, the lesions appeared as small, dark leaf spots with infrequent sporulation. Urediniospores were echinulate, mostly obovoid or elliptical and their cell walls pale-yellow to yellowish-brown. The mean width and length of urediniospores were 16.7 x 21.3 µm. No telia were observed. Applying standard rust inoculation and incubation procedures (Boshoff et al. 2022), F. pumila plants inoculated with P. nishidana isolate PREM63073 produced sporulating uredinia on the abaxial surface of leaves. Likewise, inoculation of F. carica cv. Kadota leaves with the F. pumila isolate OR835538 yielded sporulating lesions containing urediniospores typical of P. nishidana, on the lower surface. Based on both the DNA sequence data and controlled infection studies, our study confirmed the host status of F. pumila for P. nishidana. The occurrence of rust on creeping fig plants in nurseries, the evergreen status of the creeper, and thus the potential to harbor the pathogen during winter, are likely to contribute to the spread of fig rust in SA.

Wheat Stem Rust Surveillance Reveals Two New Races of Puccinia graminis f. sp. tritici in South Africa During 2016 to 2020.

Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is an important disease of wheat in South Africa (SA) and is primarily controlled using resistant cultivars. Understanding virulence diversity of Pgt is essential for successful breeding of resistant cultivars. Samples of infected wheat stems were collected across the major wheat-growing regions of SA from 2016 to 2020 to determine the pathogenic variability of Pgt isolates. Seven races were identified from 517 isolates pathotyped. The most frequently found races were 2SA104 (BPGSC + Sr9h,27,Kw) (35% frequency) and 2SA88 (TTKSF + Sr8b) (33%). Race 2SA42 (PTKSK + Sr8b), which was found in 2017, and 2SA5 (BFGSF + Sr9h), identified in 2017, are new races. The Ug99 variant race 2SA42 is similar in its virulence to 2SA107 (PTKST + Sr8b) except for avirulence to Sr24 and virulence to Sr8155B1. Race 2SA5 is closely related in its virulence to existing races that commonly infect triticale. Certain races showed limited geographical distribution. Races 2SA5, 2SA105, and 2SA108 were found only in the Western Cape, whereas 2SA107 and 2SA42 were detected only in the Free State province. The new and existing races were compared using microsatellite (SSR) marker analysis and their virulence on commercial cultivars was also determined. Seedling response of 113 wheat entries against the new races, using 2SA88, 2SA88+9h, 2SA106, and 2SA107 as controls, revealed 2SA107 as the most virulent (67 entries susceptible), followed by 2SA42 (64), 2SA106 (60), 2SA88+9h (59), 2SA88 (25), and 2SA5 (17). Thus, 2SA5 may not pose a significant threat to local wheat production. SSR genotyping revealed that 2SA5 is genetically distinct from all other SA Pgt races.

Chromosomal composition analysis and molecular marker development for the novel Ug99-resistant wheat-Thinopyrum ponticum translocation line WTT34.

KEY MESSAGE: A novel Ug99-resistant wheat-Thinopyrum ponticum translocation line was produced, its chromosomal composition was analyzed and specific markers were developed. Stem rust caused by Puccinia graminis f. sp. tritici Eriks. & E. Henn (Pgt) has seriously threatened global wheat production since Ug99 race TTKSK was first detected in Uganda in 1998. Thinopyrum ponticum is near immune to Ug99 races and may be useful for enhancing wheat disease resistance. Therefore, developing new wheat-Th. ponticum translocation lines that are resistant to Ug99 is crucial. In this study, a novel wheat-Th. ponticum translocation line, WTT34, was produced. Seedling and field evaluation revealed that WTT34 is resistant to Ug99 race PTKST. The resistance was derived from the alien parent Th. ponticum. Screening WTT34 with markers linked to Sr24, Sr25, Sr26, Sr43, and SrB resulted in the amplification of different DNA fragments from Th. ponticum, implying WTT34 carries at least one novel stem rust resistance gene. Genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization (mc-FISH), and multi-color GISH (mc-GISH) analyses indicated that WTT34 carries a T5DS·5DL-Th translocation, which was consistent with wheat660K single-nucleotide polymorphism (SNP) array results. The SNP array also uncovered a deletion event in the terminal region of chromosome 1D. Additionally, the homeology between alien segments and the wheat chromosomes 2A and 5D was confirmed. Furthermore, 51 PCR-based markers derived from the alien segments of WTT34 were developed based on specific-locus amplified fragment sequencing (SLAF-seq). These markers may enable wheat breeders to rapidly trace Th. ponticum chromosomal segments carrying Ug99 resistance gene(s).

Historical Development of the Puccinia triticina Population in South Africa.

In contrast to many other countries, the virulence and genetic diversity of the South African Puccinia triticina population before 1980 is unknown, because of the absence of regular and systematic race analysis data and viable rust cultures. Herbarium specimens housed at the National Collection of Fungi, Biosystematics Unit, Plant Health and Protection, Agricultural Research Council, Pretoria, South Africa (SA), provided the opportunity to investigate the genetic development of the population using isolates collected between 1906 and 2010. Five subpopulations that survived between 21 and 82 years in the field were found. While three of these could represent the original races that entered SA during European settlement, two appear to be recent exotic introductions into SA, most probably from other African countries. The demise of the three oldest subpopulations might be from the release of resistant wheat cultivars. The population is clonal, where new virulence develops through single step mutations and selection for virulence. Although a possible case of somatic hybridization was found, sexual reproduction appears to be absent in SA. This study confirmed the importance of annual surveys in SA and its neighboring countries for the timely detection of new virulent races that could threaten wheat production in SA.

Phenotypic and Genotypic Variation of Puccinia helianthi in South Africa.

Sunflower (Helianthus annuus L.) is the third largest grain crop by area planted in South Africa (SA). The annual yield is negatively affected by sunflower rust caused by Puccinia helianthi Schw. (Phe). Four Phe races were described in SA in the middle 1990s, but since then, no new race descriptions have been conducted. This has resulted in an information gap on the current Phe population, making it difficult to explain increased disease incidence and loss of resistance in previously resistant hybrids. To address this, 114 Phe field isolates along with 23 historic isolates were phenotyped using the international set of 11 sunflower differentials containing the R1, R2/R10, R3, R4a, R4b, R4c, R4d, R5, Pu6, and Radv resistance genes. Three new Phe races were identified, bringing the total number of South African races recorded to seven. No avirulence was detected attributable to the R1 gene, with the R4d and Radv genes remaining effective. Four main genetic lineages were detected with no obvious correlation between phenotype and genotype. The detection of three genetic lineages consisting exclusively of field isolates collected post-2006 suggested the possible recent entry of exotic introductions into SA. This, combined with the fact that one lineage consisted exclusively of the most virulent race Phe7721, confirmed a clear shift in the Phe population that could explain the increased virulence and occurrence of the disease in SA.

Microsatellite Analysis and Urediniospore Dispersal Simulations Support the Movement of Puccinia graminis f. sp. tritici from Southern Africa to Australia.

The Australian wheat stem rust (Puccinia graminis f. sp. tritici) population was shaped by the introduction of four exotic incursions into the country. It was previously hypothesized that at least two of these (races 326-1,2,3,5,6 and 194-1,2,3,5,6 first detected in 1969) had an African origin and moved across the Indian Ocean to Australia on high-altitude winds. We provide strong supportive evidence for this hypothesis by combining genetic analyses and complex atmospheric dispersion modeling. Genetic analysis of 29 Australian and South African P. graminis f. sp. tritici races using microsatellite markers confirmed the close genetic relationship between the South African and Australian populations, thereby confirming previously described phenotypic similarities. Lagrangian particle dispersion model simulations using finely resolved meteorological data showed that long distance dispersal events between southern Africa and Australia are indeed possible, albeit rare. Simulated urediniospore transmission events were most frequent from central South Africa (viable spore transmission on approximately 7% of all simulated release days) compared with other potential source regions in southern Africa. The study acts as a warning of possible future P. graminis f. sp. tritici dispersal events from southern Africa to Australia, which could include members of the Ug99 race group, emphasizing the need for continued surveillance on both continents.

Mapping a Resistance Gene to Puccinia graminis f. sp. tritici in the Bread Wheat Cultivar 'Matlabas'.

Puccinia graminis f. sp. tritici race TTKSF+ was collected from the South African wheat cultivar 'Matlabas' in 2010. F2 and F3 populations derived from a Matlabas × Line 37-07 cross segregated for a single resistance gene to race TTKSF that is avirulent to Matlabas. In screening genomic DNA bulks of susceptible or resistant F2 plants with simple sequence repeat (SSR) markers, three chromosome arm 2BS markers and one multilocus marker amplified alleles present only in the resistant bulks and Matlabas. Additional 2B-specific SSR markers, incorporating markers spanning regions containing Sr9h, SrWLR, Sr28, and Sr47, were screened in the parental lines and mapped in the F2 population. Linkage and QTL mapping showed that the gene is located between Xbarc160 in the centromeric region and Xgwm47 on the long arm of chromosome 2B. When 2B-specific SNP markers were mapped, the area of interest was delimited to a 15.3 cM region on chromosome arm 2BL, with XIWA543-HRM and Xgwm47 as flanking loci. Matlabas, Webster, and related Sr9h lines all produced a similar, low infection type to race TTKSF, but were susceptible to race TTKSF+. Phenotypic data and allelic studies suggested that stem rust resistance in Matlabas was derived from an Sr9h source.

Point Inoculation Method for Measuring Adult Plant Response of Wheat to Stripe Rust Infection.

Depending on the pathogenicity of the stripe rust fungus Puccinia striiformis f. sp. tritici, the nature of resistance in the wheat host plant, and the environment, a broad range of disease phenotypes can be expressed. Therefore, the phenotyping of partial adult plant stripe rust resistance requires reliable and repeatable procedures, especially under controlled conditions. In this study, the development of a flag leaf point inoculation method, which resulted in a 100% initial infection rate, is reported. Flag leaf inoculations were achieved by placing 6-mm antibiotic test paper discs, dipped into a urediniospore and water suspension and covered with water-proof plastic tape, on the adaxial side of leaves. Results from independent trials allowed for the statistical comparison of stripe rust lesion expansion rate in wheat entries that differ in resistance. The technique is inexpensive, reliable, and applicable to routine screening for adult plant response type, quantitative comparison of stripe rust progress, environmental influences, and pathogenicity of different isolates.

Dissection of the multigenic wheat stem rust resistance present in the Montenegrin spring wheat accession PI 362698.

BACKGROUND: Research to identify and characterize stem rust resistance genes in common wheat, Triticum aestivum, has been stimulated by the emergence of Ug99-lineage races of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), in Eastern Africa. The Montenegrin spring wheat landrace PI 362698 was identified as a source of Pgt resistance. This accession exhibits resistance to multiple Ug99-lineage and North American Pgt races at seedling and adult-plant stages. A recombinant inbred population was developed by crossing the susceptible line LMPG-6 with a single plant selection of PI 362698. A genetic map was constructed using the Illumina iSelect 90 K wheat assay and the markers csLv34, NB-LRR3, and wMAS000003 and quantitative trait locus (QTL) analysis was performed. RESULTS: QTL analysis identified five significant QTLs (α = 0.05) on chromosomes 2B, 3B, 6A, 6D, and 7A associated with wheat stem rust resistance. The QTL on chromosome 3B was identified using both field data from Kenya (Pgt Ug99-lineage races) and seedling data from Pgt race MCCF. This QTL potentially corresponds to Sr12 or a new allele of Sr12. The multi-pathogen resistance gene Sr57 located on chromosome 7D is present in PI 362698 according to the diagnostic markers csLv34 and wMAS000003, however a significant QTL was not detected at this locus. The QTLs on chromosomes 2B, 6A, and 6D were identified during seedling trials and are thought to correspond to Sr16, Sr8a, and Sr5, respectively. The QTL identified on chromosome 7A was detected using MCCF seedling data and may be Sr15 or a potentially novel allele of recently detected Ug99 resistance QTLs. CONCLUSIONS: The combination of resistance QTLs found in PI 362698 is like the resistance gene combination present in the broadly resistant cultivar Thatcher. As such, PI 362698 may not be a landrace as previously thought. PI 362698 has been crossed with North Dakota wheat germplasm for future breeding efforts. Additional work is needed to fully understand why the combination of genes present in PI 362698 and 'Thatcher' provide such durable resistance.

Yield Loss Associated with Different Levels of Stem Rust Resistance in Bread Wheat.

Puccinia graminis f. sp. tritici race Ug99 (syn. TTKSK) has been identified as a major threat to wheat production based on its broad virulence. Despite its importance, the effect of Ug99 on different types of resistance in wheat has not been thoroughly researched. In field trials conducted with P. graminis f. sp. tritici race PTKST (Ug99 race group) over 2 years, AUDPC differentiated the moderately susceptible variety SC Stallion (515) and susceptible entries SC Nduna (995) and Line 37-07 (1634) from those with adult plant resistance (APR). AUDPC of APR varieties W1406 (256), W6979 (399), and Kingbird (209) was higher than the mean of 25 recorded for the all stage resistant (ASR) variety SC Sky. In fungicide-protected and unprotected plots, race PTKST resulted in a mean yield loss of 21.3%, with susceptible Line 37-03 recording a 47.9% decrease in grain yield. Yield reduction in APR varieties reached 19.5% in W1406, whereas the ASR control SC Sky showed a mean loss of 6.4%. Although APR reduced the effects of stem rust on yield and yield components under conditions of high disease pressure, it did not provide the same protection as effective ASR.

Sources of Stem Rust Resistance in Wheat-Alien Introgression Lines.

Stem rust is one of the most devastating diseases of wheat. Widely virulent races of the pathogen in the Ug99 lineage (e.g., TTKSK) are threatening wheat production worldwide; therefore, there is an urgent need to enhance the diversity of resistance genes in the crop. The objectives of this study were to identify new sources of resistance in wheat-alien introgression derivatives from Secale cereale, Leymus mollis, L. racemosus, and Thinopyrum junceiforme, postulate genes conferring the resistance, and verify the postulated genes by use of molecular markers. From seedling tests conducted in the greenhouse, the presence of seven known stem rust resistance genes (Sr7b, Sr8a, Sr9d, Sr10, Sr31, Sr36, and SrSatu) was postulated in the wheat-alien introgression lines. More lines possessed a high level of resistance in the field compared with the number of lines that were resistant at the seedling stage. Three 2R (2D) wheat-rye substitution lines (SLU210, SLU238, and SLU239) seemed likely to possess new genes for resistance to stem rust based on their resistance pattern to 13 different stem rust races but the genes responsible could not be identified. Wheat-rye, wheat-L. racemosus, and wheat-L. mollis substitutions or translocations with single and multiple interchanges of chromosomes, in particular of the B and D chromosomes of wheat, were verified by a combination of genomic in situ hybridization and molecular markers. Thus, the present study identified novel resistance genes originating from different alien introgressions into the wheat genome of the evaluated lines. Such genes may prove useful in enhancing the diversity of stem rust resistance in wheat against widely virulent pathogen races such as those in the Ug99 lineage.

Emergence and Spread of New Races of Wheat Stem Rust Fungus: Continued Threat to Food Security and Prospects of Genetic Control.

Race Ug99 (TTKSK) of Puccinia graminis f. sp. tritici, detected in Uganda in 1998, has been recognized as a serious threat to food security because it possesses combined virulence to a large number of resistance genes found in current widely grown wheat (Triticum aestivum) varieties and germplasm, leading to its potential for rapid spread and evolution. Since its initial detection, variants of the Ug99 lineage of stem rust have been discovered in Eastern and Southern African countries, Yemen, Iran, and Egypt. To date, eight races belonging to the Ug99 lineage are known. Increased pathogen monitoring activities have led to the identification of other races in Africa and Asia with additional virulence to commercially important resistance genes. This has led to localized but severe stem rust epidemics becoming common once again in East Africa due to the breakdown of race-specific resistance gene SrTmp, which was deployed recently in the 'Digalu' and 'Robin' varieties in Ethiopia and Kenya, respectively. Enhanced research in the last decade under the umbrella of the Borlaug Global Rust Initiative has identified various race-specific resistance genes that can be utilized, preferably in combinations, to develop resistant varieties. Research and development of improved wheat germplasm with complex adult plant resistance (APR) based on multiple slow-rusting genes has also progressed. Once only the Sr2 gene was known to confer slow rusting APR; now, four more genes-Sr55, Sr56, Sr57, and Sr58-have been characterized and additional quantitative trait loci identified. Cloning of some rust resistance genes opens new perspectives on rust control in the future through the development of multiple resistance gene cassettes. However, at present, disease-surveillance-based chemical control, large-scale deployment of new varieties with multiple race-specific genes or adequate levels of APR, and reducing the cultivation of susceptible varieties in rust hot-spot areas remains the best stem rust management strategy.

Characterization of Sr9h, a wheat stem rust resistance allele effective to Ug99.

KEY MESSAGE: Wheat stem rust resistance gene SrWeb is an allele at the Sr9 locus that confers resistance to Ug99. Race TTKSK (Ug99) of Puccinia graminis f. sp. tritici, the causal fungus of stem rust, threatens global wheat production because of its broad virulence to current wheat cultivars. A recently identified Ug99 resistance gene from cultivar Webster, temporarily designated as SrWeb, mapped near the stem rust resistance gene locus Sr9. We determined that SrWeb is also present in Ug99 resistant cultivar Gabo 56 by comparative mapping and an allelism test. Analysis of resistance in a population segregating for both Sr9e and SrWeb demonstrated that SrWeb is an allele at the Sr9 locus, which subsequently was designated as Sr9h. Webster and Gabo 56 were susceptible to the Ug99-related race TTKSF+ from South Africa. Race TTKSF+ possesses unique virulence to uncharacterized Ug99 resistance in cultivar Matlabas. This result validated that resistance to Ug99 in Webster and Gabo 56 is conferred by the same gene: Sr9h. The emergence of pathogen virulence to several resistance genes that are effective to the original Ug99 race TTKSK, including Sr9h, suggests that resistance genes should be used in combinations in order to increase resistance durability.

The life cycle of Puccinia digitariae on Digitaria eriantha and Solanum species in South Africa.

Rust fungi are important plant pathogens and have been extensively studied on crops and other host plants worldwide. This study describes the heterecious life cycle of a rust fungus on Digitaria eriantha (finger grass) and the Solanum species S. lichtensteinii (large yellow bitter apple), S. campylacanthum (bitter apple), and S. melongena (eggplant) in South Africa. Following field observations, inoculation studies involving telial isolates collected from Digitaria plants produced spermogonia and aecia on S. lichtensteinii, S. campylacanthum, and S. melongena. Likewise, inoculation of finger grass with aeciospores collected from the aforementioned Solanum species produced uredinia on D. eriantha. Pennisetum glaucum (pearl millet varieties Milkstar and Okashana, as well as 17 experimental lines) and S. elaeagnifolium (silverleaf nightshade or bitter apple) were resistant to the rust isolates. Morphological descriptions and molecular phylogenetic data confirmed the identity of the rust on Digitaria as P. digitariae, herein reinstated as a species and closely related to P. penicillariae the pearl millet rust, also reinstated. Puccinia digitariae has a macrocyclic, heterecious life cycle in which teliospores overwinter on dormant D. eriantha plants. Aecia sporulate on species of Solanum during spring and early summer to provide inocula that infect new growth of Digitaria.

A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes.

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.

Tackling the re-emergence of wheat stem rust in Western Europe.

In our recent Communications Biology article, we reported the first occurrence of wheat stem rust in the UK in nearly six decades. An increased incidence of wheat stem rust in Western Europe, caused by the fungus Puccinia graminis f. sp. tritici, could signify the return of this formidable foe. As pathologists fight back against this devastating disease we outline the continuing research and strategies being employed to bridle its onslaught.

Additive Manufacturing of Devices Used for Collection and Application of Cereal Rust Urediniospores.

Optimized inoculation procedures are an important consideration in achieving repeatable plant infection when working with biotrophic rust fungi. Several plant pathology laboratories specializing in rust research employ a system where the collection and application of fungal spores are accomplished using an exchangeable gelatin capsule. Urediniospores are collected from erumpent pustules on plant surfaces into a capsule fitted to a cyclone collector controlled by a vacuum pump. By adding light mineral oil to the same capsule, the spore suspension is then sprayed onto plants by means of a dedicated atomizer (inoculator) connected to an air pressure source. Although devices are not commercially available, modern day technologies provide an opportunity to efficiently design and manufacture collectors and inoculators. Using a process called Additive Manufacturing (AM), also known as "3D printing," the bodies of a collector and inoculator were digitally designed and then laser-sintered in nylon. Depending on availability, copper or aluminum tubes were fitted to the bodies of both devices afterward to either facilitate directed collection of spores from rust pustules on plant surfaces or act as a siphon tube to deliver the spore suspension contained in the capsule. No statistical differences were found between AM and metal inoculators for spray delivery time or spore deposition per unit area. In replicated collection and inoculation tests of wheat seedlings with urediniospore bulks or single pustule collections of Puccinia triticina and P. graminis f. sp. tritici, the causal organisms of leaf rust and stem rust, consistent and satisfactory infection levels were achieved. Immersing used devices in acetone for 60 s followed by a 2 h heat treatment at 75°C produced no contaminant infection in follow-up tests.

Emergence of the Ug99 lineage of the wheat stem rust pathogen through somatic hybridisation.

Parasexuality contributes to diversity and adaptive evolution of haploid (monokaryotic) fungi. However, non-sexual genetic exchange mechanisms are not defined in dikaryotic fungi (containing two distinct haploid nuclei). Newly emerged strains of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), such as Ug99, are a major threat to global food security. Here, we provide genomics-based evidence supporting that Ug99 arose by somatic hybridisation and nuclear exchange between dikaryons. Fully haplotype-resolved genome assembly and DNA proximity analysis reveal that Ug99 shares one haploid nucleus genotype with a much older African lineage of Pgt, with no recombination or chromosome reassortment. These findings indicate that nuclear exchange between dikaryotes can generate genetic diversity and facilitate the emergence of new lineages in asexual fungal populations.

Potential for re-emergence of wheat stem rust in the United Kingdom.

Wheat stem rust, a devastating disease of wheat and barley caused by the fungal pathogen Puccinia graminis f. sp. tritici, was largely eradicated in Western Europe during the mid-to-late twentieth century. However, isolated outbreaks have occurred in recent years. Here we investigate whether a lack of resistance in modern European varieties, increased presence of its alternate host barberry and changes in climatic conditions could be facilitating its resurgence. We report the first wheat stem rust occurrence in the United Kingdom in nearly 60 years, with only 20% of UK wheat varieties resistant to this strain. Climate changes over the past 25 years also suggest increasingly conducive conditions for infection. Furthermore, we document the first occurrence in decades of P. graminis on barberry in the UK . Our data illustrate that wheat stem rust does occur in the UK and, when climatic conditions are conducive, could severely harm wheat and barley production.